High-affinity antibodies

ABSTRACT

High-affinity monoclonal antibodies, wherein the affinity is characterized by: (i) incubating first and second samples of the antibody in antigen-coated microtitre plate wells at a concentration chosen to be within the linear part of a standard curve at pH 7.2 for 1 hour at 37° C.; (ii) removing unbound antibody from both samples; (iii) incubating the first sample with PBS at pH 7.2 for 1 hour at 37° C., and reducing the pH of the second sample to pH 3 or below and incubating for 1 hour at 37° C.; (iv) removing unbound antibody from both samples; (v) incubating both samples with anti-antibody alkaline phosphatase-conjugate for 1 hour at 37° C.; (vi) removing unbound conjugate from both samples; and (vii) adding PNPP substrate to the samples, measuring the absorbance of the samples at 405 nm, and determining the amount of antibody bound to antigen, wherein the amount bound in the second sample is &gt;50% of that of the first sample.

FIELD OF THE INVENTION

[0001] This invention relates to antibodies and their therapeutic use.

BACKGROUND OF THE INVENTION

[0002] Antibodies have long been regarded as potentially powerful toolsin the treatment of cancer and other diseases. However, although therehave been some notable exceptions, this potential has not generally yetbeen realised.

[0003] This relative lack of success may be due, at least in part, tothe use of monoclonal antibodies derived from rodents, which seldom haveaffinities higher than 10⁻⁹ M. Antibodies having this level of affinityare of limited therapeutic utility, as it has proved difficult todeliver enough antibody to the target to effect useful biologicalactivity. Antibody binding to an antigen is reversible, and at theconcentrations of antibody practical for in vivo use, dissociation willbe favoured over association. In principle, it is possible to counterthe dissociation of antigen by increasing the antibody concentration.However, this may lead to unacceptable clinical side-effects and wouldalso increase the costs associated with the therapy.

SUMMARY OF THE INVENTION

[0004] The present invention is based on the realisation thatantibodies, or fragments thereof, can be produced which are“acid-resistant” and that this property is associated with high affinitybinding of an antibody for its antigen.

[0005] According to the present invention, a high-affinity antibody hasaffinity characterised by:

[0006] (i) incubating first and second samples of the antibody inantigen-coated microtitre plate wells at a concentration chosen to bewithin the linear response part of a standard curve at pH 7.2 for 1 hourat 37° C.;

[0007] (ii) removing unbound antibody from both samples;

[0008] (iii) incubating the first sample with PBS at pH 7.2 for 1 hourat 37° C., and reducing the pH of the second sample to pH 3 or below andincubating for 1 hour at 37° C.;

[0009] (iv) removing unbound antibody from both samples;

[0010] (v) incubating both samples with anti-antibodyalkaline-phosphatase conjugate for 1 hour at 37° C.;

[0011] (vi) removing unbound conjugate from both samples; and

[0012] (vii) adding PNPP substrate to the samples, measuring absorbanceof the samples at 405 nm, and determining the amount of antibody boundto antigen, wherein the amount bound in the second sample is >50% ofthat of the first sample.

[0013] Preferably, the maximum pH in step (iii) is 2.5, more preferably2.0.

[0014] Antibodies or antibody fragments with the “acid-resistant”properties are expected to favour association rather than dissociationand they therefore have longer localisation times at target sites, whichresults in a higher concentration of antibodies localised at the targetsites.

[0015] In particular, this invention relates to the production of a highaffinity single-chain Fv antibody fragment. This ScFv has particularadvantages in that it allows better targeting to a site in vivo.

DESCRIPTION OF THE DRAWING

[0016]FIG. 1 illustrates the results achieved for acid-resistance ofsheep and mouse monoclonal antibodies and single-chain Fvs with affinityto carcinoembryonic antigen at various pH values.

DESCRIPTION OF THE INVENTION

[0017] The acid-resistant monoclonal antibodies according to the presentinvention may be obtained using various techniques. For example,classical hybridoma technology can be applied, comprising the fusion ofB-lymphocytes from immunised animals secreting high-affinity antibodieswith an appropriate fusion partner. An alternative method is to purifythe mRNA from selected lymphocytes and use the technique of PCR toamplify the antibody genes required. Phage display technology and othertechniques for the display of antibody fragments may also be used toobtain the antibody genes from naive or immunised libraries afterappropriate selection procedures.

[0018] The antibody gene can be co-expressed with or otherwisechemically linked to toxins, radioisotopes or enzymes or any otherdesirable molecules to provide a fusion protein with strong bindingcharacteristics. In a further alternative, the antibodies may beproduced by transgenic animals as described in U.S. Pat. No. 5,770,429.

[0019] The antibody may be a whole antibody, comprising heavy and lightchains, and constant and variable regions. Alternatively, the antibodyis an antibody fragment, e.g. F(ab′)₂, Fab, Fv or single-chain Fvfragments, provided that at least part of the variable region is presentwhich confers the property of “acid resistance”. The antibody may alsobe an animal, chimeric or humanised antibody. A suitable method forproducing humanised antibodies is disclosed in WO-A-92/15699.

[0020] In a preferred embodiment of the invention, the antibody is asingle-chain Fv fragment. The single-chain Fv fragment comprises bothheavy chain and light chain variable regions linked by a suitablepeptide.

[0021] The antibodies of the present invention may be defined by theiracid-resistant properties, which can be characterised by an acid-washedenzyme-linked immunosorbent assay (EIA), as described above. Typicallythe A₄₀₅ value obtained by EIA will represent antibody binding of >50%for a sample at pH 3 or below, compared to the value for the sample atpH 7.2. Preferably, the A₄₀₅ value of a sample at pH 2 will representantibody binding of >60% more preferably 70% of that obtained at pH 7.2.

[0022] The animal that is subjected to immunisation is not a rodent, butis chosen to give higher affinity antibodies. Any large mammal may beused and suitable animals include rabbits, goats, cows and sheep.

[0023] An antibody of the invention may be used in therapy and may beformulated into any suitable composition with aphysiologically-acceptable excipient, diluent or carrier.

[0024] The following Examples illustrate the invention.

EXAMPLE 1

[0025] Sheep were immunised with carcinoembryonic antigen (CEA) incomplete Freund's adjuvant, then boosted three times with antigen inincomplete Freund's adjuvant. Animals were sacrificed after the finalboost and lymph nodes removed.

[0026] The lymph node cells were then washed and fused with sheepheteromyeloma fusion partner SFP3.2. Fused cells were plated out at atotal density of approximately 10⁶ per ml in medium containing HAT (LifeTechnologies). These samples were then screened for hybridomas secretinghigh-affinity antibodies to the specified antigen using both a normalEIA and an acid-washed EIA.

[0027] Standard EIA screening assays were carried out as follows:

[0028] Maxisorb assay plates (NUNC) were coated with CEA (0.4 μg/ml inphosphate-buffered saline at pH 7.2), 100 μl per well and left overnightat 4° C. The plates were then washed three times using phosphatebuffered saline at pH 7.2 with 0.01% Tween 20 detergent. Any remainingreactive sites on the plates were blocked by the addition of 200 μl perwell of 0.2% fat-free milk protein in PBS at pH 7.2 at 37° C. for ½hour. The plates were then washed in PBS as described above and 45 μl ofthe antibody samples were added to the wells of the plates. The sampleswere incubated for one hour at 37° C. and then washed as describedpreviously. Bound antibody was detected using alkalinephosphatase-conjugated donkey anti-sheep antibody (Sigma A5187 diluted1/5000 in PBS at pH 7.2 with 1% BSA). The plates were then washed and100 μl per well of PNPP (Sigma N2770) solution was added. Absorbance wasmeasured using a spectrophotometer at 405 nm with phosphate bufferedsaline as a control.

[0029] Acid-wash EIA screening assays were carried out as follows:

[0030] Coating and binding of antibody samples was as described for thestandard EIA above. However, after incubation with the antibody samples,the plates were washed and 200 μl per well of HCl (10 mM Stock solution)at pH 2 was added for one hour at 37° C. After three washes the antibodyremaining bound to antigen was detected using alkalinephosphatase-conjugated donkey anti-sheep antibody and PNPP as describedabove. In order to ensure that a proper comparison was being madebetween antibodies at different concentrations, each sample was chosento give an A₄₀₅ value of approximately 1.0 in the normal EIA (i.e. inthe linear response part of the EIA curve).

[0031] Three hybridomas (1D2, 6G11 and 6H9) secreted antibodies whichgave a greater than 50% retention of binding in the acid washed EIA, incomparison to the binding in the non-acid washed EIA.

EXAMPLE 2

[0032] A single-chain Fv fragment was produced from the hybridoma 6H9above, as follows:

[0033] mRNA was purified from the cultured hybridoma cells usingoligo-dT cellulose. Single-stranded DNA complementary to the mRNA (cDNA)was synthesized by reverse transcription. Universal primers, designedfrom the constant regions of sheep heavy and light chain antibody genes,were used in separate reverse transcription reactions to synthesise thecDNA for the antibody variable regions.

[0034] The cDNA was then amplified by the polymerase chain reaction tomake double-stranded DNA using primers designed from the heavy and lightchain variable framework sequences. Separate polymerase chain reactionswere used to amplify the heavy and light chain regions. The productswere then analysed by agarose gel electrophoresis and the DNA bandsequivalent to light and heavy chain genes were cut from the gel andpurified.

[0035] Equimolar amounts of variable heavy and light chain DNA weremixed together with an oligonucleotide linker DNA. The linker DNA codedfor the amino acid sequence (Gly₄Ser)₃ with additional nucleotidescomplementary to the 3′ end of the heavy chain variable region and the5′ end of the light chain variable region. The three DNA molecules weredenatured, annealed and extended in the first stage (without primers) ofa two-stage PCR reaction so that the fragments were joined, therebyassembling the single-chain Fv.

[0036] The single-chain Fv DNA was amplified in the second stage of thePCR using a pair of primers derived from the heavy and light chainvariable region termini with the addition of the restriction enzymerecognition sites for AlW44i and NotI. The single-chain Fv gene productwas analysed by agarose gel electrophoresis and purified. Thesingle-chain Fv was then digested with the restriction enzymes AlW44iand NotI and cloned into an expression vector. The vector was then usedto transform E. coli HB 2151, and protein expression was allowed tooccur. The vector was designed so as to include a hexa-histidine tag atthe COOH terminus of the SFv. The single-chain Fv was purified usingnickel-chelate affinity chromatography and analysed by SDS-PAGE. Theamino acid sequence for the heavy chain variable region and the lightchain variable region is disclosed in SEQ ID Nos. 2 and 4, respectively.An acid-wash EIA was also carried out to determine the acid-resistantproperties of the single-chain Fv.

[0037] Acid-wash EIA was carried out as follows:

[0038] Carcinoembryonic antigen (CEA)-coated microtitre plates wereprepared as described previously. Single-chain Fv samples (6H9) werediluted to a range of concentrations between 1 ng/ml and 100 ng/ml inPBS at pH 7.2 containing 1% bovine serum albumin (BSA). 100 μl sampleswere added to the microtitre plate wells and incubated for 1 hour at 37°C. The plates were then washed, 200 μl per well of citrate added, andthe plates incubated for 1 hour at 37° C. In this case, the acidpreparations were made using a stock solution of 100 mM citrate dilutedto pH values of 4.0, 3.5, 3.0, 2.5 and 2.0 in the reaction mixture. PBSat pH 7.2 was used as a reference control. The plates were then washedand 100 μl per well of mouse anti-tetra-histidine antibody (Qiagen) (100ng/ml diluted in PBS at pH 7.2 with 1% BSA) added and incubated for 1hour at 37° C. After plate washing the samples were incubated for 1 hourat 37° C. With 100 μl per well of goat anti-mouse alkaline phosphataseconjugate (Sigma A3688 diluted 1/1000 in PBS with 1% BSA at pH 7.2). Theplates were then washed, treated with PNPP as described previously andthe absorbance measured using a spectrophotometer at 405 nm.

[0039] As a control for acid resistance, sFv samples were incubated withPBS at pH 7.2 to generate an EIA response curve for the SFv samples. Inthe linear region, a concentration of 10-20 ng/ml of the SFv sample gavean absorbance (A₄₀₅) of 1.0-1.5 and was therefore used to determine theamount of antibody bound in the acid washed samples as a percentage ofthe amount bound in the reference sample.

[0040] The acid-resistant properties of the 6H9 whole antibody and the6H9 single-chain Fv were compared with that for the mouse-derivedanti-carcinoembryonic antigen whole antibody, A5B7 and the single-chainFv MFE. The results are shown in FIG. 1, with the antigen-binding of themouse-derived antibodies being substantially reduced at pH 3.5 and lessthan 5% at pH 2.5. In contrast, the 6H9 antibodies retain >70% antigenat pH 3.5, >60% at pH 2.5 and >50% at pH 2.0.

1 4 1 363 DNA Artificial Sequence CDS (1)..(363) Description ofArtificial SequenceAntibody Fragment 1 cag gtg cag ctg cag gag tcg ggaccc agc ctg gtg aag ccc tca cag 48 Gln Val Gln Leu Gln Glu Ser Gly ProSer Leu Val Lys Pro Ser Gln 1 5 10 15 acc ctc tcc ctc acc tgc acg gtctct gga ttc tca tta acc aag tat 96 Thr Leu Ser Leu Thr Cys Thr Val SerGly Phe Ser Leu Thr Lys Tyr 20 25 30 ggt gtt agt tgg gtc cgc cag gct ccagga aag gcg ctt gag tgg cta 144 Gly Val Ser Trp Val Arg Gln Ala Pro GlyLys Ala Leu Glu Trp Leu 35 40 45 ggt ggt gtg tcc agt ggt gca cta aca gcctat aac aca gcc cta cag 192 Gly Gly Val Ser Ser Gly Ala Leu Thr Ala TyrAsn Thr Ala Leu Gln 50 55 60 tcc cga ctc agc gtc acc agg gac acc tcc aagagc caa ttc tcc ctg 240 Ser Arg Leu Ser Val Thr Arg Asp Thr Ser Lys SerGln Phe Ser Leu 65 70 75 80 tca ctg agc agc gtg act act gag gac acg gccatt tac tac tgt gcg 288 Ser Leu Ser Ser Val Thr Thr Glu Asp Thr Ala IleTyr Tyr Cys Ala 85 90 95 aaa tct gtc aat ggt gac agt gtt cct tat ggt ttggac tac tgg agc 336 Lys Ser Val Asn Gly Asp Ser Val Pro Tyr Gly Leu AspTyr Trp Ser 100 105 110 cca gga ctc cta ctc acc gtc tcc tca 363 Pro GlyLeu Leu Leu Thr Val Ser Ser 115 120 2 121 PRT Artificial SequenceDescription of Artificial Sequence Antibody Fragment 2 Gln Val Gln LeuGln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu SerLeu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Lys Tyr 20 25 30 Gly Val SerTrp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Gly ValSer Ser Gly Ala Leu Thr Ala Tyr Asn Thr Ala Leu Gln 50 55 60 Ser Arg LeuSer Val Thr Arg Asp Thr Ser Lys Ser Gln Phe Ser Leu 65 70 75 80 Ser LeuSer Ser Val Thr Thr Glu Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Lys SerVal Asn Gly Asp Ser Val Pro Tyr Gly Leu Asp Tyr Trp Ser 100 105 110 ProGly Leu Leu Leu Thr Val Ser Ser 115 120 3 333 DNA Artificial SequenceCDS (1)..(333) Description of Artificial sequence Antibody Fragment 3cag gat gtg ctg act cag ccg tcc tcc gtg tct ggg tcc ctg ggc cag 48 GlnAsp Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln 1 5 10 15agg gtc tcc atc acc tgc tct gga agc agc agc aac att gga ggt aat 96 ArgVal Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn 20 25 30 gcttat gtg ggc tgg tac caa cag gtc cca gga tca gcc ccc aga ctc 144 Ala TyrVal Gly Trp Tyr Gln Gln Val Pro Gly Ser Ala Pro Arg Leu 35 40 45 ctc atcagt gct aca acc gat cga gcc tcg ggg atc ccc gac cga ttc 192 Leu Ile SerAla Thr Thr Asp Arg Ala Ser Gly Ile Pro Asp Arg Phe 50 55 60 tcc ggc tccagg tct ggg aac aca gcc acc ctg acc atc agc tcg ctc 240 Ser Gly Ser ArgSer Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 cag gct gaggac gag gcc gat tat tac tgt gca tcg tat caa agt act 288 Gln Ala Glu AspGlu Ala Asp Tyr Tyr Cys Ala Ser Tyr Gln Ser Thr 85 90 95 tac agt ggt gttttc ggc agc ggg acc agg ctg acc gtc ctg ggt 333 Tyr Ser Gly Val Phe GlySer Gly Thr Arg Leu Thr Val Leu Gly 100 105 110 4 111 PRT ArtificialSequence Description of Artificial Sequence Antibody 4 Gln Asp Val LeuThr Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln 1 5 10 15 Arg Val SerIle Thr Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn 20 25 30 Ala Tyr ValGly Trp Tyr Gln Gln Val Pro Gly Ser Ala Pro Arg Leu 35 40 45 Leu Ile SerAla Thr Thr Asp Arg Ala Ser Gly Ile Pro Asp Arg Phe 50 55 60 Ser Gly SerArg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln AlaGlu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Tyr Gln Ser Thr 85 90 95 Tyr SerGly Val Phe Gly Ser Gly Thr Arg Leu Thr Val Leu Gly 100 105 110

I claim:
 1. A polynucleotide molecule encoding a high-affinitymonoclonal antibody, wherein the affinity of said antibody ischaracterisable by: (i) incubating first and second samples of theantibody in antigen-coated microtitre plate wells at a concentrationchosen to be within the linear part of a standard curve at pH 7.2 for 1hour at 37° C.; (ii) removing unbound antibody from both samples; (iii)incubating the first sample with PBS at pH 7.2 for 1 hour at 37° C., andreducing the pH of the second sample to pH 3 or below and incubating for1 hour at 37° C.; (iv) removing unbound antibody from both samples; (v)incubating both samples with anti-antibody alkalinephosphatase-conjugate for 1 hour at 37° C.; (vi) removing unboundconjugate from both samples; and (vii) adding PNPP substrate to thesamples, measuring the absorbance of the samples at 405 nm, anddetermining the amount of antibody bound to antigen, wherein the amountbound in the second sample is >50% of that of the first sample, whereinsaid antibody has a heavy chain variable region comprising the aminoacid sequence defined in SEQ ID No. 2 and a light chain variable regioncomprising the amino acid sequence defined in SEQ ID No. 4, or a variantthereof; and wherein the polynucleotide comprises a nucleotide sequencedefined in SEQ ID Nos. 1 and 3, or a variant thereof.
 2. A cloningvehicle comprising a polynucleotide molecule encoding a high-affinitymonoclonal antibody, wherein the affinity of said antibody ischaracterisable by: (i) incubating first and second samples of theantibody in antigen-coated microtitre plate wells at a concentrationchosen to be within the linear part of a standard curve at pH 7.2 for 1hour at 37° C.; (ii) removing unbound antibody from both samples; (iii)incubating the first sample with PBS at pH 7.2 for 1 hour at 37° C., andreducing the pH of the second sample to pH 3 or below and incubating for1 hour at 37° C.; (iv) removing unbound antibody from both samples; (v)incubating both samples with anti-antibody alkalinephosphatase-conjugate for 1 hour at 37° C.; (vi) removing unboundconjugate from both samples; and (vii) adding PNPP substrate to thesamples, measuring the absorbance of the samples at 405 nm, anddetermining the amount of antibody bound to antigen, wherein the amountbound in the second sample is >50% of that of the first sample; whereinsaid antibody has a heavy chain variable region comprising the aminoacid sequence defined in SEQ ID No. 2 and a light chain variable regioncomprising the amino acid sequence defined in SEQ ID No. 4, or a variantthereof; and wherein the polynucleotide comprises a nucleotide sequencedefined in SEQ ID Nos. 1 and 3, or a variant thereof.